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ORIGINAL ARTICLE
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Comparative scores of ABO and Rh hemagglutination reactions


1 Department of Physiology, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Pimpri, Pune, India
2 Sable Pathology Laboratory, Warora, Chandrapur, Maharashtra, India

Date of Submission16-Oct-2020
Date of Decision16-Oct-2020
Date of Acceptance19-Jun-2021

Correspondence Address:
Prashant Devidas Khuje,
Department of Physiology, DR.D.Y.P.M.C, Pimpri, Pune, 411018, F-104, Wing-H, Eden-II, Mahindra Royale, CTS 6020, Pimpri, Pune - 411 018, Maharashtra
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/mjdrdypu.mjdrdypu_574_20

  Abstract 


Background: Blood group determination is done by the slide test and tube test. Grading/scoring of hemagglutination reactions is performed through the naked eye and microscopy to detect spurious (false positive and negative) reactions. Objectives: In our observational study, comparative scoring of ABO and Rh hem-agglutination reactions was done based on degree of hemolysis and agglutination through the macroscopic and microscopic technique. Materials and Methods: The determination of blood group of 209 participants was done by the porcelain slide method using commercially prepared antisera (manufactured by TULIP DIAGNOSTICS (P) LTD.) containing anti-A, anti-B, and anti-D monoclonal antibodies followed by scoring/grading based on degree of hemolysis and red blood cell clumping. The data were statistically analyzed using the Chi-square test and Z-test of proportion. Results: ABO hemagglutination reactions showed significantly higher score ([A-Macro Z = 7.153, Micro Z = 4.65] [B-Macro Z = 11.17, Micro Z = 21]) (Z >2.5, P < 0.001) than Rh hemagglutination reactions. B hemagglutination reactions showed insignificantly higher score (B-Macro Z = 1.29, Micro Z = 1.816) (Z <1.95, P < 0.05) than A hemagglutination reaction. Conclusion: ABO hemagglutination reactions showed significantly higher score than Rh hemagglutination reactions as ABO system agglutinin are pentavalent (immunoglobulin M) binding 10 agglutinogens while that of Rh system are bivalent binding 4 agglutinogens. B hemagglutination reactions showed insignificantly higher score than A hem-agglutination reaction which required further evaluation.

Keywords: ABO RH scores



How to cite this URL:
Khuje PD, Karandikar MS, Sable BU. Comparative scores of ABO and Rh hemagglutination reactions. Med J DY Patil Vidyapeeth [Epub ahead of print] [cited 2022 Dec 7]. Available from: https://www.mjdrdypv.org/preprintarticle.asp?id=338028




  Introduction Top


Red blood cell (RBC) surface membrane contains a variety of genetically determined blood group agglutinogens (BGs antigens), while plasma contains noncorrespondent agglutinins (antibody).[1],[2],[3] However, the ABO and Rh BG system has fundamental importance in transfusions. In clinical laboratories, it is the standard procedure to test for BGs A (containing only A antigens), B (containing only B antigens), AB (having both A- and B antigens), O (neither A nor B antigens), and Rh (giving information about the presence or absence of Rh antigens).[4],[5],[6] During blood group determination, the red cells having surface agglutinogens are made to react with the commercially available antisera-containing known agglutinins. The presence or absence of clumping (agglutination) and hemolysis of red cells determines the blood group according to the ABO and Rh system. Grading/scoring of agglutination reactions is performed using the slide or tube sample.[1],[7]

Scoring assists in detecting the presence of more than one antibody, dosage effect of antigens, abnormal weak reactions, and spurious (false positive) reactions.[8]

The present study was planned to examine the scores of ABO and Rh blood group hem-agglutination reactions by the naked eye (NE) examination and simple microscopy according to the degree of hemolysis and agglutination followed by comparing the ABO and Rh system hemagglutination reactions.


  Materials and Methods Top


After receiving approval from the Institutional Ethics Committee, determination of blood group of 209 participants was done by the porcelain slide method followed by scoring after getting participants' informed consent.

Inclusion criteria

Fresh capillary blood samples from normal healthy male and female adults of age ranged between 18 and 25 years were included in the study.

Exclusion criteria

Stored blood, clotted blood samples, and blood samples with any in vitro anticoagulants and unwilling participants were excluded from the study.

Six pits arranged in two rows of three each on a dry and clean porcelain tile were labeled as A (ABO antigen A), C (control) and B (ABO antigen B) on top row, and D (Rh antigen D) and S (red cell suspension [RCS]) on the 1st and 2nd pits of the second row, respectively.

A drop of commercially prepared antisera (manufactured by TULIP DIAGNOSTICS (P) LTD.) containing monoclonal Anti-A, Anti-B, and Anti-D antibodies (directed against corresponding blood group antigens) and citrated normal saline were poured in pits labeled A, B, D, and C pits, respectively, with separate droppers for each fluid. After pricking the finger under all aseptic precautions, two drops of blood were poured and mixed with 4 drops of citrated normal saline contained in pit “S” to get a homogeneous RCS.[8],[9],[10],[11]

A drop of this suspension was transferred to each of the pit A, C, B, and D. Mixing of the contents was done with separate glass rod for each pit solutions.

The reactions were read by single and same observer after 6–10 min with intermittent blowing at 37°C.[1],[11] Pit C acted as the control to check contamination.

The nature and type of blood group antigen and antibody decide the formation of agglutinate and degree of hemolysis. ABO and Rh agglutination reactions were then scored by NE[11],[12] as follows after blowing the solution in the pit for 5 s.

Grade 4: – single solid agglutinate with/without clear background

Grade 3:– 2 to 4 large agglutinates with/without clear background

Grade 2:– 5 to10 medium-sized agglutinates with/without clear background

Grade 1:– >10 small aggregates with/without clear background

Grade 0:– Granular turbid suspension.

After NE examination, scoring by microscopy (M) was done by same observer through wet slide preparation of the pit solution placed on the glass slide followed by placing a cover slip and was then observed and interpreted by same observer under low power magnification.[12],[13]

Grade 4: – Single large aggregate per field with/without free RBCs

Grade 3: – 2 to 4 large aggregates per field with/without free RBCs

Grade 2: – 5 to 10 medium-sized aggregates per field with/without free RBCs

Grade1: – >10 small aggregates per field in the background of RBCs

Grade 0: – No aggregates in the background of free RBCs

Negative: – Freely scattered RBCs with no aggregates.

Statistical analysis

The data were statistically analyzed by the Chi-square test and Z-test of proportion.


  Results Top


ABO hemagglutination reactions showed significantly higher score ([A-Macro Z = 7.153, Micro Z = 4.65] [B-Macro Z = 11.17, Micro Z = 21]) (Z > 2.5, P < 0.001) than Rh hemagglutination reactions. B hemagglutination reactions showed insignificantly higher score (B-Macro Z = 1.29, Micro Z = 1.816) (Z <1.95, P < 0.05) than A hemagglutination reaction [Table 1]. Eleven negative reactions detected by macroscopic examination were confirmed as true positive by microscopy.
Table 1: Analysis of scores of ABO and Rh haem-agglutination reactions

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  Discussion Top


In the present study, porcelain slide was used for blood group typing and macroscopic scoring followed by microscopy. Plain slides were for blood group determination.[1],[13]

Hemagglutination reactions were graded using the slide samples. They were graded as single large aggregate (Grade 4+), 2–3 large to medium aggregates (Grade 3+), 4–7 medium to small masses (Grade 2+), and many small aggregates in the background of free red cells (Grade 1+).[1],[14]

Grading of ABO and Rh agglutination reactions was done macroscopically using the test tube samples. NE examinations of reactions within the tubes after shaking described the grades as Grade (4+) with 1 big clump, Grade (3+) with 2 or 3 clumps, Grade (2+) with many small clumps with clear supernatant, Grade (1+) with many small clumps with turbid supernatant and Grade (Zero or H) with smooth suspension.[8],[12]

Hemagglutination reactions interpretation was done based on microscopic examination using centrifuged test tube blood sample. These grades were described as Grade (4+): solid agglutinate with clear background, Grade (3+): with breaking of solid agglutinate into several large agglutinates with clear background, Grade (2+) with many medium-sized agglutinates with clear background but no free RBCs, Grade (1+) with many small aggregates/clumps barely visible macroscopically with a turbid background and many free RBCs, and Grade (0) or negative with no clumps with free RBCs.[12],[14]

In the present study, comparison between ABO and Rh system hemagglutination reactions was done along with intra-ABO hemagglutination (A and B) reaction. ABO hemagglutination reactions showed significantly higher score than Rh hemagglutination reactions. B hemagglutination reactions showed insignificantly higher score than A hemagglutination reaction.

ABO group system agglutinin is pentavalent immunoglobulin M (IgM) binding 10 agglutinogens (forming comparatively larger agglutinates) while that of Rh agglutinin are bivalent-binding four agglutinogens. Therefore, ABO blood group system agglutinates showed higher scores than Rh blood group system agglutinate. B hemagglutination reactions showed higher scores than A.


  Conclusion Top


ABO hemagglutination reactions showed significantly higher score than Rh hemagglutination reactions as ABO agglutinin are pentavalent (IgM) binding 10 agglutinogens while that of Rh system are bivalent-binding four agglutinogens. B hemagglutination reactions showed insignificantly higher score than A hemagglutination reaction which require further evaluation.

Limitations

Modern-day methods/techniques such as microplate, column gel configuration, nucleic acid amplification, etc., should have been done to determine the specific BGs antigens and to quantify hemagglutination reactions.

Financial support and sponsorship

This study was financially supported by Dr. D. Y. Patil Medical College, Hospital and Research Center, Pimpri, Pune - 411 018, Maharashtra, India.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Ghai GL. A Textbook of Practical Physiology Section 1. Chapter: Blood Grouping. 7th ed. Jaypee Brothers Medical Publishers (P) Ltd New Delhi, India 2007:p. 103-16.  Back to cited text no. 1
    
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Landsteiner K. The anti-fermentative-lytic and agglutinating effect of blood serum and the lymph; Central Sheet bacterial; Parasite Infection 1900;27:357-62.  Back to cited text no. 2
    
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Landsteiner K. Agglutination phenomenon in normal human blood. Vienna Klin Wochenschr 1901;14:1132-4.  Back to cited text no. 3
    
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Liu Z, Liu M, Mercado T, Illoh O, Davey R. Extended blood group molecular typing and next-generation sequencing. Transfus Med Rev 2014;28:177-86.  Back to cited text no. 4
    
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Rowley M, Milkins C. Laboratory aspects of blood transfusion. In: Lewis SM, Barbara JB, Imelda B, editors. Dacie and Lewis Practical Haematology. 10th ed. Philadelphia, PA, USA: Churchill Livingstone; 2006. p. 523-54.  Back to cited text no. 5
    
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Mujahid A, Dickert FL. Blood group typing: from classical strategies to the application of synthetic antibodies generated by molecular imprinting. Sensors (Basel) 2015;16:51.  Back to cited text no. 6
    
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Virginia Vengelen-Tyler Technical Manual of the American Association of Blood Banks. 13th ed. 1117 North 19th Street, Suite-600. Arlington. V.A- 22209: Glenbrook Road, Bethesda, Maryland US:1999:20814-749.  Back to cited text no. 7
    
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Hoffbrand Victor A, Daniel C, Edward GD, Antony G. Red cell immune-haematology: Introduction. In: Postgraduate 42 Haematology. 6th ed., Ch. 14. Red cell immunohaematology: Wiley Blackwell Publications; 2010 43. Ltd; Hoboken, New Jersey, US, 2010:p. 226-71.  Back to cited text no. 8
    
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Richard LG, Foerster J, Lukens J, skevas FP, Greer JP, Rodgers GM. Wintrobe's Clinical Hematology. 10th ed. Baltomore, Maryland, US,1245 Williams and Wilkins, Lippincott Williams and Wilkins; 2014;46:p. 817-75.  Back to cited text no. 9
    
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Mannessier L, Roubinet F, Chiaroni J. Problem-solving in immuno-hematology: Direct compatibility laboratory test. Transfus Clin Biol 2011;8:481-4.  Back to cited text no. 10
    
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Laboratory Manual of Haematology, Clinical and Experimental Physiology, Chap -Determination of blood groups; M.U.H.S Nashik, MS, India. 2011: p;17-8.  Back to cited text no. 11
    
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Daniels G, Bromilow I. Essential Guide to Blood Groups. 2nd ed. Wiley Blackwell Publications; Hoboken, New Jersey, US. 2011:p. 1-73.  Back to cited text no. 12
    
13.
Harvey KC, David AJ; Mollison's Blood transfusion in clinical Medicine, Chap:-3 Red cell immunology; and Chap. 4 ABO, Lewis and P blood groups; 11th edition, Blackwell Publising Ltd; Hoboken, New Jersey, US; 2008; pp 48-113.  Back to cited text no. 13
    
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Quinley D, Eva; Immunohematology: Principles and Practice; 3rd edition; Wolters Kluwer/Lippincott Williams & Wilkins publications; Philadelphia, US; 2011:p.54-147.  Back to cited text no. 14
    



 
 
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